MHC presentation of peptide has broad importance, as shown by the range of disease conditions influenced by MHC molecules, e.g. infections, tumors, transplantation rejection, and autoimmunity. Cell surface major histocompatibility complex (MHC) class I molecules present tumor- or pathogen-specific peptides to T cells, which respond by lysing malignant or infected cells. Self-MHC class I/peptide complexes are required for the primary selection of the host's repertoire of cytolytic T lymphocytes. In addition, the number of surface MHC class I molecules on target cells influences their recognition by natural killer cells. For all these reasons, cell surface expression of peptide-bearing MHC class I molecules is of immunological importance. Before egress to the cell surface, MHC class I molecules receive peptides within a complex of proteins in the endoplasmic reticulum. In the peptide-loading complex, the protein calreticulin binds to the MHC class I heavy chain, and its presence in the complex is up-regulated by tapasin. Tapasin binds to the MHC class I heavy chain, recruits ERp57 and the transporter associated with antigen processing (TAP), and stabilizes TAP. Although tapasin serves as a critical factor in the control of MHC class I surface expression and peptide presentation, the mechanisms whereby it regulates MHC class I assembly are poorly understood. A more complete understanding of the importance of MHC class I peptide-loading complex interactions involving tapasin would contribute to the creation of strategies to influence peptide presentation for many therapeutic purposes. The objective of this project is to define the mechanisms by which antigen presentation by MHC class I molecules is regulated. Our central hypothesis is that specific interactions of tapasin with other assembly complex proteins regulate the sequence and affinity of MHC class I-binding peptides, the stability of MHC class I/p2m heterodimers prior to final peptide binding, and ultimately cytotoxic T lymphocyte selection and response. We propose these Aims: 1) to determine the role of peptide-loading complex interactions in the stabilization of complete MHC molecules and in the regulation of the MHC class I peptide repertoire, 2) to define the effect of assembly complex interactions on the stability of open MHC class I/p2m heterodimers, and 3) to characterize the impact of peptide-loading complex interactions on T cell recognition, response, and development, using in vitro and in vivo systems. At the completion of this project, it is our expectation that we will have defined contributions of the peptide-loading complex involving tapasin to the regulation of the MHC class I-binding peptide repertoire, MHC class I stabilization, and T cell function.